Osteoarthritis or degenerative joint disease is a slowly progressive, irreversible, often monoarticular disease characterized by pain and loss of function (Mankin and Brandt, Pathogenesis of Osteoarthritis in xe2x80x9cTextbook of Rheumatologyxe2x80x9d, Kelly, et al., (eds.) 3rd edition, W. B. Saunders Co., Philadelphia, pp.14699-111471) and Dean, Arth. Rheum. 20 (Suppl. 2):2 (1991)). The underlying cause of the pain and debilitation is the cartilage degradation that occurs as a result of the disease. A typical end-stage clinical picture includes complete erosion of the weight-bearing articular cartilage, requiring total joint replacement.
A class of inhibitors of protein kinase C are hymenialdisines and hymenialdisine analogues (Nambi et al., WO 93/16703). For example, debromohymenialdisine, inhibits protein kinase C gamma (108% at 100 xcexcm), protein kinase C alpha (97% at 100 xcexcm), protein kinase C beta (95% at 100 xcexcm), as well as Ca2+-calmodulin dependent protein kinase II, (94% a 100 xcexcm). Hymenialdisine and hymenialdisine analogues contain a pyrroloazepine ring system, shown below, along with a numbering system for the ring atoms: 
Analogues of hymenialdisine also have a five membered, nitrogen-containing heterocyclic ring which is bonded to the four position of the pyrroloazepine ring system. Examples of hymenialdisines and analogues thereof which have been shown to inhibit cartilage degradation in the bovine cartilage explant assay include Z-debromohymenialdisine, E-debromohymenialdisine and Z-hymenialdisine (Experientia., 44:86 (1988) and Pettit, et al., Can. J. Chem., 68:1621 (990).
Z-hymenialdisine or Z-debromohymenialdisine are represented by the formula: 
Z-debromohymenialdisine also slows the progression of osteoarthritis in animals. The use of hymenialdisines and analogues thereof for the treatment of osteoarthritis is disclosed in Chipman and Faulkner, U.S. Ser. No. 08/472,902 filed Jun. 7, 1995, now U.S. Pat. No. 5,591,740, issued on Jan. 7, 1997, the entire teachings of which are incorporated herein by reference.
Currently, other than the hymenialdisines and analogues thereof, discussed above, there is no known, demonstrated therapeutic approach available that will slow the clinical progression of osteoarthritis, although steroids and non-steroidal anti-inflammatory drugs are used to ameliorate the pain and inflammation associated with the disease. Consequently, there is a need for new therapeutics which slow the joint degeneration caused by osteoarthritis.
It has now been found that tyrosine kinase inhibitors significantly reduce or prevent cartilage degradation in chondrocytes. Specifically, the tyrosine kinase inhibitors genistein, herbimycin A, 4,5-dianilinophthalimide (DAPH), tyrphostin AG 82 and tyrphostin AG 556 slow interleukin-1 (IL-1) induced degradation of extracellular matrix by chondrocytes in cell culture (Examples 1 and 7). Herbimycin A and tyrphostin AG 82 also reduce cartilage degradation in a bovine cartilage explant assay (Examples 2 and 3). Protein tyrosine kinase inhibitors have also been shown to inhibit IL-1 induced increases in stromelysin mRNA levels (Example 4) and IL-1 induced increases in prostromelysin protein levels (Example 5). Based on these discoveries, methods of treating individuals with osteoarthritis and methods of inhibiting cartilage degradation in individuals are disclosed.
One embodiment of the present invention is a method of treating an individual or animal with osteoarthritis. The method comprises administering a therapeutically effective amount of a protein tyrosine kinase inhibitor to the individual or animal. Another embodiment of the present invention is a method of inhibiting or preventing cartilage degradation in an individual or animal. The method comprises administering a therapeutically effective amount of a tyrosine kinase inhibitor to the individual or animal.
The disclosed method of treatment inhibits the cartilage degradation associated with the osteoarthritis. Treatments presently used for osteoarthritis only alleviate the symptoms of the disease, for example the pain and inflammation that result from joint deterioration. Therefore, the disclosed treatment for osteoarthritis has the advantage over presently used methods of treatment in that the disclosed method can slow or arrest the progression of the disease rather than merely alleviate its symptoms.
Protein tyrosine kinases (PTKs) occur as membrane-bound receptors or cytoplasmic proteins. They are involved in regulating a wide variety of cellular functions, including cytokine responses, antigen-dependent immune responses, cellular transformation by RNA viruses, oncogenesis, cell cycle, and modification of cell morphology. PTKs regulate these functions by activating, directly or indirectly, intracellular signalling pathways, including Ras, phosphatidylinositol 3 kinase (PI3K), phospholipase C-xcex3 (PLC-xcex3) and mitogen-activated pathway (MAP). It has now been found that PTKs also regulate cellular functions which result in the cartilage degradation associated with osteoarthritis.
Activation of PTKs results in auto-phosphorylation of a tyrosine residue in the protein tyrosine kinase. Auto-phosphorylation of PTKs facilitates the interaction of protein substrates with the active site and results in the phosphorylation of tyrosine residues in the protein substrates. Protein substrates of PTKs are generally cytosolic signalling molecules whose function is turned off or on as a result of phosphorylation. Activation of protein substrates by PTKs can cause a cascade of intracellular reactions resulting in the activation of other proteins or previously unexpressed or underexpressed genes. This cascade of events is referred to as a signalling pathway, which regulates cellular functions, including the cellular functions discussed above.
Because PTKs regulate cellular functions which cause the cartilage degradation associated which osteoarthritis, the progression of the disease can be slowed or arrested by inhibiting PTKs. As used herein, xe2x80x9cinhibiting a PTKxe2x80x9d refers to blocking the signal transduction pathway whereby an activated PTK regulates a cellular function. In the present invention, a PTK inhibitor is used which blocks a signal transduction pathway in which an activated PTK regulates one or more cellular functions which result in cartilage degradation. Included are PTK inhibitors which reduce cartilage degradation in IL-1 activated chondrocytes in cell culture, which downregulate matrix metalloproteinase (MMP) and/or aggrecanase mRNA levels in IL-1 activated chondrocytes in cell culture or which downregulate MMP and/or aggrecanase protein levels in chondrocytes in cell culture.
A PTK inhibitor includes a small organic molecule or polypeptide which blocks a PTK regulated signaling pathway, as discussed above. As used herein, a PTK inhibitor can act by a number of different mechanisms. Preferably, the PTK inhibitor can act by inhibiting the initial autophosphorylation event, discussed above. Alternatively, the PTK inhibitor can act by inhibiting the phosphorylation of the protein substrate, for example, by competing with the protein substrate or ATP for binding with the PTK. A PTK can also act by more than one of these mechanisms.
As used herein, a PTK inhibitor can act by other mechanisms. For example, compounds which prevent binding of activating molecules (e.g. growth factors) to receptor PTKs, either by blocking the receptor (e.g. a receptor antagonist) or by binding with the activating molecule itself. Alternatively, a PTK inhibitor can act by blocking one of the biochemical reactions in the cascade of reactions initiated by activation of the PTK. For example, as noted above, activation of a PTK can result in the activation of the Ras, phosphatidylinositol 3 kinase (PI3K), phospholipase C-xcex3 (PLCxcex3) and mitogen-activated pathway (MAP). Agents which can block any one of these pathways following their initiation by PTK activation can also downregulate cellular functions controlled by the respective PTK.
PTK inhibitors suitable for use in the method of treatments disclosed herein include PTK inhibitors which are natural products. Examples include quercetin, genistein, lavendustin A, erbstatin, herbimycin A, rapamycin, piceatannol and lavendustin B. The chemical structures of these compounds are provided in the 1995 CALBIOCHEM(copyright) Signal Transduction Catalog, (pages 143-153) (hereinafter the xe2x80x9cCALBIOCHEM(copyright) Catalogxe2x80x9d).
PTK inhibitors suitable for use in the method of treatments disclosed herein also include synthetic PTK inhibitors. Synthetic PTK inhibitors are disclosed in the 1995 CALBIOCHEM(copyright) Catalog (pages 143-153) and in Levitzki and Gazit, Science 267:1782 (1995).
In one embodiment, the synthetic PTK inhibitor used in the method of treatment comprises a compound represented by Structural Formula (I): 
wherein m is one or two; R1 is xe2x80x94H, xe2x80x94OH or xe2x80x94OMe; R2 is xe2x80x94H or xe2x80x94CN; and RI is xe2x80x94H, xe2x80x94NO2, halogen or an organic radical chosen such that the compound represented by Structural Formula (I) inhibits PTKs. Examples of suitable organic radicals include xe2x80x94CN, xe2x80x94COxe2x80x94NH2, xe2x80x94CSxe2x80x94NH2, xe2x80x94COxe2x80x94NHR10, xe2x80x94CSxe2x80x94NHR10, phenyl, substituted phenyl, substituted heteroaryl and heteroaryl (e.g. pyrimidyl, pyridinyl). Suitable substituents for a substituted alkyl or alkenyl group include xe2x80x94NH2, xe2x80x94NO2, halogen, xe2x80x94CN, xe2x80x94COxe2x80x94NH2, xe2x80x94CSxe2x80x94NH2, xe2x80x94COxe2x80x94NHR10, xe2x80x94CSxe2x80x94NHR10, phenyl substituted phenyl, substituted heteroaryl and heteroaryl. R10 is a substituted or unsubstituted C1 to about C8 straight or branched chain alkyl or alkenyl group. Suitable substituents for a phenyl or heteroaryl group include halogen, xe2x80x94NO2, xe2x80x94CN and C1-C4 straight or branched chain alkyl. A substituted phenyl, alkyl or alkenyl group can have more than one substituent.
Examples of compounds represented by Structural Formula (I) include dihydroxynitrostyrenetyrphostin AG18, tyrphostin AG82, tyrphostin AG99, tyrphostin AG213, tyrphostin AG308, tyrphostin AG494, tyrphostin AG555, 3,4-dihydroxy-cis-cinnamonitrile, tyrphostin AG825, tyrphostin AG765, tyrphostin A48, tyrphostin A51, tyrphostin B42, tyrphostin B44(xe2x88x92), tyrphostin B46, tyrphostin B48, tyrphostin B50(+) and tyrphostin B56. The structures of these compounds are disclosed in Levitzki and Gazit and/or in the 1995 CALBIOCHEM(copyright) Catalog on pages 143-153. Preferably, the PTK inhibitor,represented by Structural Formula (I) comprises a 3,4-dihydroxy-cis-cinnamonitrile moiety, i.e., m is 2, R1 is xe2x80x94H and R2 is xe2x80x94CN.
In another embodiment, the synthetic PTK inhibitor used in the method of treatment comprises a dianilinophthalimide moiety represented by Structural Formula (II): 
wherein R3-R6 are each independently selected from the group consisting of xe2x80x94H, xe2x80x94Cl, xe2x80x94OH and xe2x80x94OMe. Examples includes 3,4-dianilinophthalimide and 2,5-dianilinophthalimide. The structure of these compounds are disclosed in Levitzki and Gazit.
In another embodiment, the synthetic PTK inhibitor used in the methods of treatment disclosed herein is a compound which comprises a quinoline moiety or an isoquinoline moiety and is represented by Structural Formula (III): 
wherein n is one, two or three; RIII is an organic radical chosen such that the compound represented by Structural Formula (III) inhibits PTKs. Examples of suitable organic radicals include xe2x80x94COxe2x80x94NH2, xe2x80x94CSxe2x80x94NH2, xe2x80x94COxe2x80x94NHR10, xe2x80x94CSxe2x80x94NHR10, substituted alkyl and substituted alkenyl. R10, substituted alkyl and substituted alkenyl are as defined above for Structural Formula (I). Examples of compounds which are PTK inhibitors and which comprise an isoqunioline moiety include compounds represented by Structural Formula (IV): 
In another embodiment, the synthetic PTK inhibitor used in the method of treatment is a compound comprising a quinazoline moiety and is represented by Structural Formula (V): 
wherein n is one, two or three; RV is an organic radical chosen so that the compound represented by Structural Formula (V) is a PTK inhibitor. RV can be, for example, xe2x80x94NHR11, xe2x80x94OR11, SR11, wherein RV is a phenyl group, substituted phenyl group, substituted heteroaryl group or heteroaryl group (e.g. pyrimidyl or pyridinyl) optionally substituted with one or more substituents selected from the group consisting of halogen, xe2x80x94OH, xe2x80x94OMe, xe2x80x94NH2, xe2x80x94CN and xe2x80x94NO2. Tyrphostin AG1478 is one example of a compound represented by Structural Formula (V). The structure of this compound is disclosed in Levitzki and Gazit. See also Barker, European Patent Application 0520722 (1992), Fry et al., Science 265:1093 (1994), and Osherov and Levitizki, Eur. J Biochem. 225:1047 (1994). The entire teachings of these references are incorporated by reference into this application.
In another embodiment, the synthetic PTK inhibitor used in the method of treatment is a compound comprising a flavone or isoflavone moiety and is represented by Structural Formula (VI): 
wherein n is one, two or three; RVI is an organic radical chosen so that the compound represented by Structural Formula (VI) is a PTK inhibitor. RVI can be, for example, a phenyl group or heteroaryl group (e.g. pyrimidyl or pyridinyl) substituted with one or more substituents selected from the group consisting of halogen, xe2x80x94OH, xe2x80x94OMe, xe2x80x94NH2, xe2x80x94CN and xe2x80x94NO2. Aminogenistein is one example of a PTK inhibitor represented by Structural Formula (VI). The structure of this compound is disclosed in the 1995 CALBIOCHEM(copyright) Catalog.
In another embodiment, the PTK inhibitor is represented by Structural Formula (VII): 
wherein m, R1 and R2 are as described above for Structural Formula (I); p is an integer from one to about eight, preferably from one to about four.
In another embodiment the PTK inhibitor is a tryphostin. Tryphostins are defined in Mazunder et al., Biochemistry 34:15111 (1995), the entire teachings of which are incorporated into this application by reference.
Other examples of PTK inhibitors include tyrphostin AG10, tyrphostin AG17, tyrphostin AG825, tyrphostin AG789, tyrphostin AG1112, tyrphostin AG 370, tyrphostin AG 879, Bis-tyrphostin, 5-amino-N-(2,5-dihydroxybenzyl)methyl salicylate, 2,5-dihydroxymethylcinnamate, HNMPA-(AM)3, RG-13022, RG-14620 and ST638. Compounds which inhibit the catalytic site of tyrosine kinases include the Ca2+, antagonists chlorpromazine, imipramine and dibucaine (End et al., Res. Commun. Chem. Pathol. Pharmacol. 107:670 (1987), flavanoids (Hagiwara et al., Biochem. Pharmacol. 37:2987 (1987), 4-hydroxycinnamides (Shiraishi et al., Biochem. Biophys. Res. Commun. 147:322 (1987) and xcex1-cyanocinnamides (Shiraishi et al., Chem. Pharm Bull. 36:974 (1988). For structures of these compounds, see also Levitzki and Gazit or the 1995 CALBIOCHEM(copyright) Catalog.
It is to be understood that many modifications to the Structural Formulas I-VII and to the compounds listed above can be made which result in analogs which are also PTK inhibitors. Such modifications include replacing a phenolic hydroxyl group with an xe2x80x94H, a lower alkyl group (e.g., a C1-C4 straight or branched chain alkyl group), xe2x80x94Cl, xe2x80x94OCH3, or xe2x80x94NH2 or adding a xe2x80x94Cl, xe2x80x94OCH3 or xe2x80x94NH2 group to a phenol or resorcinol ring. Analogs such as those described above are included within the meaning of the term xe2x80x9cPTK inhibitorxe2x80x9d, and can be identified by in vitro assays by their ability, for example, to inhibit IL-1 stimulated cartilage degradation in chondrocytes in culture, for example the assay described in Example 1.
The method of the present invention can be used to treat individuals, i.e. humans, or animals with osteoarthritis. It can also be used to slow or prevent cartilage degradation in individuals or animals with a condition which causes cartilage degradation. Animals which can be treated with the method include dogs, cats, guinea pigs, horses, farm animals and the like.
A xe2x80x9ctherapeutically effect amountxe2x80x9d of a protein tyrosine kinase inhibitor is the quantity of inhibitor which, after being administered to an individual or animal with osteoarthritis, brings about an amelioration of the disease processes associated with osteoarthritis without causing unacceptable side-effects. xe2x80x9cAmeliorating the disease processes associated with osteoarthritisxe2x80x9d can include lowering the amount of active matrix metalloproteinase in the individual, e.g. by inhibiting a matrix metalloproteinase, by preventing transcription of a gene which encodes a matrix metalloproteinase, by preventing the synthesis and/or secretion of a matrix metalloproteinase or by preventing interleukin-1 upregulation of matrix metalloproteinase activity. Alternatively, it can also include slowing, arresting or reversing the degradation and loss of function typically observed in a joint afflicted with osteoarthritis, e.g. by reducing the rate of cartilage degradation in the joint. xe2x80x9cAmeliorating the disease processes associated with osteoarthritisxe2x80x9d can also include a lessening of the pain and inflammation associated with osteoarthritis.
The skilled artisan will be able to determine the amount of inhibitor which is to be administered to a human or animal. The amount of PTK inhibitor that is administered to an individual or animal will depend on a number of factors including the general health, size, age, and sex of the individual or animal and the route of administration. It will also depend on the degree, location and severity of the individual""s or animal""s osteoarthritis or cartilage degradation. One of ordinary skill in the art will be able to determine the precise dosage according to these and other factors. Typically, between about 0.1 mg per day and about 1000 mg per day are administered to the individual. Preferably, between about 0.1 mg per day and about 100 mg per day are administered to the individual, more preferably between about 1 mg per day and about 30 mg per day. The amount of PTK inhibitor administered to an animal will also depend on the type of animal.
The inhibitor can be administered intraarticularly (for example by injection) into a joint with cartilage degradation caused by osteoarthritis. Intra-articular injection has the advantage that the inhibitor is localized to the site of injection and that the concentration of inhibitor in other parts of the body is reduced. This is particularly advantageous in reducing undesirable side-effects when the protein tyrosine kinase inhibitor used to treat osteoarthritis or reduce cartilage degradation is non-specific and inhibits other protein kinases. Other modes of parenteral administration which can be used include systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.
In a preferred embodiment, the inhibitor can be administered orally, for example, in capsules, suspensions or tablets. Alternatively, the inhibitor can be administered topically near the joint with cartilage degradation caused by osteoarthritis.
The PTK inhibitor can be administered to the individual or animal in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition for treating osteoarthritis. Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the PTK inhibitor. Standard pharmaceutical formulation techniques may be employed such as those described in Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceutical carriers for intraarticular and other parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank""s solution, Ringer""s-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al., xe2x80x9cControlled Release of Biological Active Agentsxe2x80x9d, John Wiley and Sons, 1986). Suitable carriers for topical administration include commercially available inert gels, liquids supplemented with albumin, methyl cellulose or a collagen matrix. Typical of such formulation are ointments, creams and gels. Preferred carriers for topical administration are those which facilitate penetration of the skin by the PTK inhibitor.
The PTK inhibitor can also be administered as at least one physiologically acceptable salt, such as, the hydrochloride salt, the hydrobromide salt and acetic acid salt.
In another embodiment of the present invention the composition, in addition to the inhibitor, additionally comprises one or more pharmacologically active agent. Osteoarthritis is characterized by pain in the afflicted joints. Consequently, it can be advantageous to administer the PTK inhibitor with an analgesic or other pain-killing medication. Suitable analgesics include acetyl salicylic acid, acetominophen, and the like.
Osteoarthritis can be characterized by inflammation in the afflicted joints. Consequently, it may also be advantageous to administer the PTK inhibitor together with an anti-inflammatory agent such as a non-steroidal anti-inflammatory drug or steroid (e.g. triamcinolone, amcinodide, and the like). Osteoarthritis is also characterized by over-activity of matrix metalloproteinase enzymes. Consequently, it may also advantageous to co-administer the PTK inhibitor with a matrix metalloproteinase inhibitor.